Lai M, Thomason D B, Weisbrodt N W
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77030.
Am J Physiol. 1990 Jan;258(1 Pt 2):R39-43. doi: 10.1152/ajpregu.1990.258.1.R39.
In this study, messenger RNAs (mRNAs) for actin isoforms were assessed in longitudinal smooth muscle from the ileum of unoperated rats and from rats that had undergone bypass of the middle 70% of the small intestine. The plasmid clone pGEM 10C, which contains a DNA insert complementary to the 3' untranslated region and the region of mRNA that codes for the synthesis of alpha-smooth muscle actin protein, was used to synthesize two riboprobes. One probe, complementary to the coding region of the insert, hybridizes to most, if not all, actin isoform mRNAs. The second probe, complementary to the 3' untranslated region of the insert, hybridizes only to alpha-smooth muscle actin mRNA. RNA was isolated from animals 4 to 5 days after operation, size fractionated by denaturing gel electrophoresis, transferred to nylon membranes, and exposed to the two 32P-labeled riboprobes. Both probes hybridized to RNA of about 1.3 kilobases long. Longitudinal muscle from both groups of animals contained alpha-smooth muscle actin mRNA as well as mRNA for other actin isoforms. Dot blots of varying amounts of RNA were hybridized to the riboprobes to determine the proportions of actin mRNAs. The content and concentration of mRNAs for all actins, and of mRNA for alpha-smooth muscle actin, were significantly greater in muscle from the functioning ileum of bypassed animals 4-5 days after the operation. Thus the operation induces a rapid, specific activation of these contractile protein genes.
在本研究中，对未手术大鼠回肠以及经历了小肠中段70%旁路手术的大鼠回肠的纵行平滑肌中的肌动蛋白异构体信使核糖核酸（mRNA）进行了评估。质粒克隆pGEM 10C含有与3'非翻译区以及编码α-平滑肌肌动蛋白合成的mRNA区域互补的DNA插入片段，用于合成两种核糖探针。一种探针与插入片段的编码区互补，可与大多数（即便不是全部）肌动蛋白异构体mRNA杂交。第二种探针与插入片段的3'非翻译区互补，仅与α-平滑肌肌动蛋白mRNA杂交。在术后4至5天从动物体内分离RNA，通过变性凝胶电泳进行大小分级，转移至尼龙膜上，并与两种32P标记的核糖探针杂交。两种探针均与约1.3千碱基长的RNA杂交。两组动物的纵行肌均含有α-平滑肌肌动蛋白mRNA以及其他肌动蛋白异构体的mRNA。将不同量RNA的点杂交与核糖探针杂交，以确定肌动蛋白mRNA的比例。术后4至5天，旁路手术动物功能正常的回肠肌肉中，所有肌动蛋白mRNA以及α-平滑肌肌动蛋白mRNA的含量和浓度均显著更高。因此，该手术可诱导这些收缩蛋白基因迅速、特异性地激活。
